PERGAMON
Phytochemistry 40 "0888# 24Ð30
E}ect of ca}eine on shot!hole borer beetle "Xyleborus fornicatus#
of tea "Camellia sinensis#
Priyadarshine Hewavitharanage0\ Subodhi Karunaratne\ N[ Savitri Kumar
Department of Chemistry\ University of Peradeniya\ Peradeniya\ Sri Lanka
Received 09 August 0887
Abstract
Ca}eine was found to inhibit oviposition in shot!hole borer beetle "Xyleborus fornicatus# of tea "Camellia sinensis# in laboratory
culture media[ Ca}eine also delayed the appearance of the di}erent developmental stages in the life cycle\ but apparently did not
have a lethal e}ect on the beetle[ The inhibitory e}ect of ca}eine on the beetle was partially reduced in the presence of the polyphenol
tannic acid[ Þ 0888 Published by Elsevier Science Ltd[ All rights reserved[
Keywords] Xyleborus fornicatus^ Shot!hole borer beetle^ Camellia sinensis^ Monacrosporium ambrosium^ Fungus^ Ca}eine^ Oviposition^ Polyphenols^
Tannic acid
0[ Introduction Thus interactions between ca}eine and other types of
naturally occurring molecules may well determine the
The purine alkaloid ca}eine\ the major alkaloid found relative susceptibility of cultivars to attack by insect pests
in tea "Camellia sinensis#\ was found to inhibit the growth and microbial pathogens[ A preliminary study was car!
of the ambrosia fungus "Monacrosporium ambrosium#\ ried out to determine whether the e}ect\ if any\ of ca}eine
the fungal symbiote of the shot!hole borer beetle on SHB could be altered in the presence of a polyphenolic
"Xyleborus fornicatus# "Kumar\ Hewavitharanage\ + compound such as tannic acid[
Adikaram\ 0884#[ We suggested that the accumulation of
ca}eine in tea stems after beetle attack\ could be a plant
defence strategy[ This paper is a report of in vitro experi!
1[ Results and discussion
ments carried out using laboratory culture media con!
taining ca}eine\ to determine the e}ect\ if any\ of ca}eine
The shot!hole borer beetle is found in symbiotic associ!
on the shot!hole borer "SHB#[
ation with the ambrosia fungus "M[ ambrosium# within
Phenols and polyphenols are important constituents of
galleries made by the beetle in tea stems "Gadd + Loos\
tea plants[ Phenolic compounds play a central role in
0836#[ The fungus is probably the sterol source neces!
plantÐherbivore interactions and also in~uence the inter!
sary for the development of the larval stages
action of herbivores with microbial symbiotes[ Ca}eine
"Wickremasinghe\ Perera\ + Perera\ 0865#[ We have
has been reported to form stable complexes with poly!
reported "Kumar et al[\ 0884# that ca}eine inhibited the
phenols such as potassium chlorogenate "Gorter\ 0896^
growth of the ambrosia fungus and that there was
Gorter\ 0897# and complex formation was attributed to
accumulation of ca}eine after beetle attack[ Previous
dipole!induced dipole interactions which may be of
studies have shown that ca}eine has a toxic e}ect on
physiological signi_cance "Booth\ Boyland\ + Orr\ 0843#[
insects at concentrations found in plants "Frischknecht\
Complex formation between chlorogenic acid and
Ulmer!Dufek\ + Baumann\ 0875# and it has been sug!
ca}eine reduced the toxicity of ca}eine in rabbits
gested that ca}eine may be an endogenous substance
"Gerhadt\ 0828# and was also found to inhibit the action
produced by plants to discourage insect feeding
of ca}eine on frog muscle "Straub + Domejoz\ 0830#[
"Nathanson\ 0873#[ Hence the e}ect of ca}eine on SHB
could be of interest[
Two separate experiments involving ca}eine were car!
0
Present address] Plant Physiology Division\ Coconut Research Insti!
ried out[ In the _rst experiment\ the beetle was cultured
tute\ Lunumila\ Sri Lanka[
Corresponding author[ in the laboratory with varying amounts of ca}eine in the
9920!8311:88:, ! see front matter Þ 0888 Published by Elsevier Science Ltd[ All rights reserved[
PII] S9920!8311"87#99509!3
25 P[ Hewavitharanage et al[ : Phytochemistry 40 "0888# 24Ð30
culture medium[ These experiments were carried out to in the presence of the tea bark extract and the fungus was
obtain information regarding the concentrations of observed after 7 and 04 days when the medium contained
ca}eine at which normal growth of the fungus was evi! tea bark extract together with 099 and 199 ppm of ca}eine
dent and the e}ect of ca}eine on the development of the respectively[ A similar e}ect was seen when tea bark
SHB[ In the second study the number of days required extract was added to the medium containing 499\ 0999
to observe each stage of the life cycle was determined[ and 1999 ppm of ca}eine respectively "Table 1#[ Only
During this study the e}ect of tannic acid in the culture slight growth of the fungus was observed after 15 and 29
medium\ on appearance of di}erent developmental stages days\ respectively\ in the tubes with 0999 and 1999 ppm
in the life cycle of SHB\ was also determined[ of ca}eine[ The fungus did not grow at all in tubes with
4999 ppm of ca}eine "diet types 2 and 3# con_rming our
1[0[ Effect of different concentrations of caffeine on SHB earlier _ndings that ca}eine has an inhibitory e}ect on
the fungus at this concentration "Kumar et al[\ 0884#[
Twenty!four diet tubes were prepared for each con! The larval stages appeared after 07 days and the _rst
centration of ca}eine "49\ 099\ 199\ 499\ 0999\ 1999 and female from the second generation emerged after 29 days
4999 ppm# and these consisted of six replicates of four from the two sets of control tubes 0 and 1[ Emerging
types of diet media "Sivapalan\ 0865#[ Thereafter\ one females were not observed in any of the diet media 2 and
female beetle per tube was added and the development 3[ The total number of emerging females was 66 in the
of the fungus and larvae were monitored[ control tubes 1 "with tea bark extract# while it was only
It was observed that galleries were made on all four 29 in control tubes 0 "without tea bark extract#[ Therefore
types of diet tubes[ The growth of M[ ambrosium started tea bark extract apparently contains additional sub!
from the second day of inoculation in the control tubes stances which are favorable to the development of the
with diet types 0 and 1[ The presence of ca}eine in the beetles[ It is likely that secondary metabolites present in
diet medium delayed the appearance of the fungus which the tea bark either act as oviposition stimulants or signal
was observed after 5\ 09 and 08 days in media containing the presence of a suitable host medium[
49\ 099 and 199 ppm respectively of ca}eine[ The fungus Although the fungus was absent in tubes containing
appeared only after 19Ð13 days in the tubes with 499 ppm 4999 ppm of ca}eine\ some of the introduced beetles were
of ca}eine "Table 0#[ However the growth pattern of the alive up to about 29 days[ Hence ca}eine does not have
fungus was normal in the presence of 49Ð499 ppm of a lethal e}ect on SHB[ However these beetles did not
ca}eine and occurred to a similar extent in all these tubes[ deposit eggs[ In tubes with 0999 and 1999 ppm of ca}eine\
The inhibitory e}ect of ca}eine was somewhat mitigated fungal growth was slight and some beetles were alive for
Table 0[ E}ect of ca}eine on development of Ambrosia fun`us and shot!hole borer
Ca}eine ABC D E
Composition of the medium Diet type "ppm# galleries fungus larval stages adult females total females
Control medium "CM# 0 Ð 0 1 07 29 29
CMÅšextracta 1 Ð 0 1 072966
CMÅšextractÅšca}eine 2 49 0 5 Ð Ð 9
CMÅšca}eine 3 49 0 5 Ð Ð 9
CMÅšextractÅšca}eine 2 099 0 7 Ð Ð 9
CMÅšca}eine 3 099 0 09 Ð Ð 9
CMÅšextractÅšca}eine 2 199 1 04 Ð Ð 9
CMÅšca}eine 3 199 1 08 Ð Ð 9
CMÅšextractÅšca}eine 2 499 1 19 Ð Ð 9
CMÅšca}eine 3 499 1 13 Ð Ð 9
CMÅšextractÅšca}eine 2 0999 2 15 Ð Ð 9
CMÅšca}eine 3 0999 2 18 Ð Ð 9
CMÅšextractÅšca}eine 2 1999 2 29 Ð Ð 9
CMÅšca}eine 3 1999 2 21 Ð Ð 9
CMÅšextractÅšca}eine 2 4999 2 none Ð Ð 9
CMÅšca}eine 3 4999 2 none Ð Ð 9
a
Extract refers to tea bark extract[
A is the number of clays for appearance of galleries[
B is the number of days for appearance of fungus[
C is the number of days before observing larval stages[
D is the number of days before emergence of females[
E is the total number of emerging females[
P[ Hewavitharanage et al[ : Phytochemistry 40 "0888# 24Ð30 26
about 29 days\ but eggs were not deposited[ Eggs were brown in color# "vi# appearance of mature beetles "dark
also not observed in tubes containing 499\ 199\ 099 and brown in color# and "vii# emergence of female beetles[
49 ppm of ca}eine\ though fungal growth appeared The total number of females which emerged from each
normal[ In the tubes with 49 ppm of ca}eine some of the medium in each generation was also counted[ Obser!
beetles remained alive up to 59 days[ Eggs\ larval stages vations were made on six replicates of each of the four
and emerging females were not observed in all the tubes di}erent culture media[
containing ca}eine "diet media 2 and 3#[ This experiment
con_rmed the inhibitory e}ect of ca}eine on the fungus 1[2[ Observations made
and also indicated that ca}eine has an inhibitory e}ect
on oviposition of the beetle[ 0[ Galleries were observed in 1Ð2 days in A "Fig[ 0a#
According to our results\ oviposition is inhibited even whereas they were observed after one day in both C
when the concentration of ca}eine is 49Ð099 ppm[ and D[ Therefore gallery formation takes place at a
Nathanson "0873# reported that larvae of the tobacco signi_cantly faster rate in the media containing tannic
hornworm Manduca sexta were killed in a nutrient med! acid[ Galleries were observed after 2Ð3 days in the
ium containing 9[2) "2\999 ppm# ca}eine and in Cal! tube containing the medium B "only ca}eine#[
losobruchus chinensis ca}eine caused nearly 099) 1[ The fungus was observed in A as well as in C and D
sterility at a ca}eine concentration of 0[4) "Rizvi\ after 1 days\ but in B this observation was made after
Pandey\ Mukerji\ + Mathur\ 0879#[ SHB infests and thri! 4 days "Fig[ 0b#[ Therefore ca}eine signi_cantly slows
ves in tea stems which contain 249Ð649 ppm of ca}eine down the development of the fungus while tannic acid
"dry weight# "Karunaratne + Kumar#[ Our experiments did not a}ect fungal growth "D#\ and reduced the
also showed that the highest number of beetles emerged ca}eine e}ect "C#[
from the control tubes 0 with tea bark extract in which the 2[ Eggs were observed _rst in A and C "four days# and
ca}eine concentration is 0049Ð0219 ppm "Karunaratne + only after 01 days in B "Fig[ 0c#[ Observation of eggs
Kumar#[ The in vivo tolerance of SHB beetle to such high was di.cult because the eggs are laid at the bottom of
concentrations of ca}eine may be due to the presence of the gallery and are sometimes covered by the beetle[
other phytochemicals which are able to overcome the In D "tannic acid# eggs were seen after 6 days\ i[e[ later
inhibitory e}ect of ca}eine[ It is also possible that ca}eine than in C "3Ð4 days# containing both ca}eine and
may be in a biologically inactive or bound form in healthy tannic acid[ Therefore the combination of tannic acid
tea stems and is released after attack by the beetle[ For and ca}eine was more favorable for the development
example\ association between ca}eine and naturally of the beetle than the medium containing only tannic
occurring molecules such as polyphenols in aqueous acid "D# or only ca}eine "B#[
media could remove ca}eine from the medium[ Gorter 3[ Larvae were visible in A after about 09Ð00 days\
"0896\ 0897# reported that only a small fraction of ca}eine whereas they were observed only after 05Ð07 days in
can be extracted with chloroform from the crystalline B\ C and D "Fig 0d#[ The larvae tended to move out
complex formed between ca}eine and potassium chlo! of the galleries and into the space between the medium
rogenate[ It is also known that such complex formation and the walls of the tube in search of food because the
results in aggregates which are precipitated from solution fungal mycelia were observed to grow well on the
"McManus\ Davis\ Haslam\ + Lilley\ 0870#[ Therefore outer surface of the medium[ Therefore the presence
polyphenols\ which are important constituents of tea of either ca}eine or tannic acid\ as well as a com!
could be of signi_cance in reducing the inhibitory e}ect bination of the two delayed the appearance of larvae[
of ca}eine on SHB in tea stems[ 4[ The immature beetles are light brown in color and
their movements are fast[ The immature beetles were
1[1[ Effect of caffeine and tannic acid on shot!hole borer slowest to appear in B "17 days#\ while in C and D this
beetles observation was made after about 11 days "Fig[ 0e#[
Immature beetles in A were observed after about 12Ð
In these experiments the control medium A contained 13 days[
sucrose\ yeast\ casein\ Wesson|s salt\ agar\ cellulose and 5[ The mature beetles were _rst observed in A\ C\ then
distilled water[ The diet medium B contained the control in D "about 29 days# and later "about 24 days# in B
medium with ca}eine "099 ppm#\ while medium C was "Fig[ 0f #[ These beetles were dark brown in color and
composed of control medium AÅštannic acidÅšca}eine were very mobile[ The mature beetles took a few days
"099 ppm each#[ Medium D contained control medium to move towards the cotton plugs and to emerge from
AÅštannic acid "099 ppm#[ the galleries[ Both immature and mature beetles were
Observations were made on the time "number of days# slowest to appear in B\ the medium containing only
taken before "i# formation of the _rst gallery\ "ii# obser! ca}eine[
vation of the fungus\ "iii# egg formation\ "iv# appearance 6[ The total number of beetles which emerged from B
of larvae\ "v# appearance of immature beetles "light was very small "3# while 49Ð44 beetles emerged from
27 P[ Hewavitharanage et al[ : Phytochemistry 40 "0888# 24Ð30
Fig[ 0[ Number of days for appearance of di}erent developmental stages in the life cycle of Shot!hole borer "SHB#[
A "Fig[ 0h#[ The number which emerged from C "i[e[ number of beetles from the control medium during the
medium containing both ca}eine and tannic acid# was second experiment[ The thin layer of medium may well
larger "07# than from D "00#\ but much less than from be more favorable for the construction of galleries and\
A[ the survival and development of the larval stages[ This
may also account for the emergence of a very small num!
Some di}erences were noted in the results obtained from ber of beetles from the ca}eine containing medium B in
the two experiments[ In the _rst experiment larval stages the second experiment while none emerged from this
were observed only after 07 days in the control medium medium during the _rst experiment[
whereas in the second this observation was made in 09Ð Our results suggest that ca}eine in laboratory culture
00 days[ The di}erence may be attributed to the fact that media has an inhibitory e}ect on oviposition[ Oviposition
the diet medium was placed in glass tubes in the _rst is the _rst stage in the insectÐplant relationship at which
experiment\ whereas in the second the diet medium was the plant may show resistance "Stanley\ 0854#[ Thus plant
placed as a thin layer between the inner wall of a boiling resistance to the onion maggot Hylemya antiqua\ was
tube and the outer wall of a test tube placed in the center found to be entirely due to resistance to oviposition
of the boiling tube[ The latter set!up made observation "Perron + Jasmin\ 0852#[ Plant secondary metabolites
of the developmental stages easier and more accurate[ play an important role in ovipositional behavior[ The
The di}erence in the set!up of the medium may also tobacco hornworm Protoparce sexta\ oviposits on the
account for the observation of larval stages in the medium Solanaceae and eggs are deposited only in response to an
B "containing ca}eine# and the emergence of a larger alcohol soluble substance "Yamamoto + Fraenkel\ 0859#[
P[ Hewavitharanage et al[ : Phytochemistry 40 "0888# 24Ð30 28
Fig[ 0*continued[
Therefore the inhibition of oviposition in laboratory nolic content of the less susceptible clone TRI 1912
media containing ca}eine may well be due to the absence "Abeysinghe\ Kumar\ + Ratnayake#[ It is likely that the
of another host plant chemical which acts as an oviposition content and the composition of phenolics in tea stems
stimulant in tea stems[ may have a bearing on di}erences in susceptibility of
According to our results ca}eine signi_cantly delayed certain tea clones to infestation by SHB[
the appearance of each developmental stage in the life
cycle of the beetle[ Thus eggs were observed after 01 days 2[ Experimental
in medium B\ whereas eggs were seen in 2Ð3 days in both
the control medium A and the medium C containing 2[0[ Collection of beetles
both ca}eine and tannic acid[ The _rst instar larvae were
observed after 09 days in A\ whereas larvae were observed The shot!hole borer beetle of tea is sensitive to both
only after 07 days in B\ the medium containing ca}eine[ heat and light\ and the activity of this beetle varies
The appearance of mature beetles and the number of depending on the daily temperature[ The beetle is most
days taken for the beetles to emerge were also delayed in active from noon until about 2[29 p[m[ at Peradeniya
medium B[ where the temperature is around 14216>C throughout
Observations on the media containing tannic acid sug! the year[ The beetles are active between 1[29 and 2[99
gest that an interaction\ possibly complexation\ between p[m at Talawakelle\ where the temperature is lower and
tannic acid and ca}eine reduces the bioavailability of ~uctuates between 05Ð19>C throughout the year[ There!
ca}eine[ We have observed that the phenolic content of fore all observations were made at 1[29Ð2[99 p[m[ each
the susceptible clone TRI 1914 is greater than the phe! day when the beetles were presumed to be most active[
39 P[ Hewavitharanage et al[ : Phytochemistry 40 "0888# 24Ð30
2[1[ Laboratory culture of beetles maintained at room temperature "15219># in the dark[
Observations regarding gallery construction\ eggs\ num!
{Mother beetles| used to start the _rst generation of ber of larvae\ pupae and emerging female beetles were
the laboratory culture were collected from beetle infested made daily for 61 days[
bushes of the tea clone TRI 1914 "tea clone reported to Twenty!four tubes were incubated for each ca}eine
be the most susceptible to attack by SHB# at the TRI! concentration^ six tubes "control medium with tea bark
substation in Hantana[ Only healthy adult female beetles extract#\ six tubes "control medium#\ six tubes "control
"X[ fornicatus# which were about to emerge were medium tea bark extractÅšca}eine# and six tubes "control
collected[ The beetles were surface sterilized with 1) medium with ca}eine#[
NaOCl "1Ð2 min#\ washed with distilled water and intro!
duced into tubes containing the culture medium prepared 2[3[ Effect of caffeine and tannic acid on shot!hole borer
under sterile conditions[ The composition of the culture beetles
media used are given below[ Each medium was prepared
2[3[0[ Preparation of culture tubes
by mixing all the constituents thoroughly in a mortar[
An equal quantity of the medium was poured into each
of six boiling tubes[ A test tube was placed in the center
2[2[ Effect of different caffeine concentration on shot! of the boiling tube so that there was a thin layer of
hole borer beetles medium between the walls of the inner test tube and the
outer boiling tube[ The boiling tubes were sealed with
2[2[0[ Preparation of culture media
cotton plugs and aluminium foil\ and autoclaved[ The
Beetles were reared in the laboratory in a diet medium
culture tubes were kept for 2Ð3 days before use because
"149 ml# "Sivapalan\ 0865# composed of sucrose "3[6 g#\
excess moisture in the tubes or on the upper surface of
casein "2[0 g#\ yeast extract "2[0 g#\ Wesson|s salt mixture
the medium was found to restrict the movement of the
"9[3 g#\ cellulose powder "49[9 g#\ _ne agar "01[4 g# and
beetles[ The tubes were kept undisturbed in the dark
H1O "014 ml#[ Penicillin was added to the culture media
"13 h# at 14216>C "Sivapalan\ 0865#[
to prevent bacterial contamination[ Bacterial growth
results in a medium which is sticky and the beetle is
2[3[1[ Composition of the media
unable to move about freely under these conditions[
The basal medium " medium A# used to culture the
Dietary ingredients other than agar\ were made into a
beetles was composed "mg# of sucrose "0\398#\ yeast "818#\
paste with H1O "14 ml#[ Agar was soaked in H1O "099
casein "818#\ Wesson|s salt "007#\ agar "2\638#\ cellulose
ml#\ added to the paste and mixed well[ Tea bark extract
"06\999# and distilled water "64 ml#[ Medium A served as
was made from freshly peeled bark "14 g# of healthy
control\ and was supplemented with 099 ppm ca}eine
pencil thick tea stems\ the preferential site of attack
"medium B#\ 099 ppm ca}eine and 099 ppm tannic acid
"Danthanarayana\ 0862#\ from the clone TRI 1914\ cut
"C#\ or with 099 ppm tannic acid "D#[
in to small pieces\ blended with water "014 ml# and
_ltered[ The tea bark extract "014 ml# was added to the
Acknowledgments
agar paste and mixed well[ Water alone "014 ml# was
added to prepare the control diet medium[
The authors thank the Tea Research Institute "TRI#
Four types of diet media were prepared[ "0# Control
at Talawakelle and the TRI substation at Hantane for
medium\ "1# control mediumÅštea bark extract\ "2# con!
cooperation and assistance\ the Sri Lanka Council for
trol mediumÅštea bark extractÅšca}eine and "3# control
Agricultural Research Policy "CARP# for sponsoring the
mediumÅšca}eine[
project\ the International Seminar in Chemistry "IPICS#\
Ca}eine was dissolved in distilled H1O "09 ml#\ mixed
Uppsala University\ Sweden and the Swedish Agency for
well with cellulose powder and incorporated in varying
Research Cooperation "SAREC# for research grants[
concentrations "4999\ 1999\ 0999\ 499\ 199\ 099\ 49 ppm#
into the culture medium and the volume of H1O used to
dissolve ca}eine was reduced from the volume of water
References
used to soak the agar[ Ca}eine was incorporated into the
diet medium before autoclaving[
Abeysinghe\ S[\ Kumar\ N[ S[\ + Ratnayake\ R[ M[ S[ K[ Unpublished
The diet medium "04 ml# was placed in glass:boiling
results[
tubes "049×04 mm# which were then plugged with sur!
Booth\ J[\ Boyland\ E[ + Orr\ S[ F[ D[\ "0843#[ J[ Chem[ Soc[\ 487[
gical cotton[ The diet tubes were autoclaved and allowed Danthanarayana\ W[ "0862#[ Ent[ Exp[ Appl[\ 05\ 294[
Frischknecht\ P[ M[\ Ulmer!Dufek\ J[\ Baumann\ T[ W[ "0875#[ Phyto!
to cool to room temperature[ Penicillin "499 ml\ 49
chemistry\ 14\ 502[
units:ml# was added and the tubes were kept at room
Gadd\ C[ H[\ Loos\ C[ A[ "0836#[ Trans[ Br[ Mycol[ Soc[\ 20\ 02[
temperature for 13 h[ One freshly emerged young female
Gerhadt\ H[ "0828#[ Arch[ Exptl[ Pathol[ Pharmakol[\ 080\ 585[
of X[ fornicatus was added into each tube under sterile
Gorter\ K[ "0896#[ Justus Liebig|s Ann[ Chem[\ 247\ 216[
conditions\ the tubes were plugged immediately\ and Gorter\ K[ "0879#[ Justus Liebig|s Ann[ Chem[\ 248\ 106[
P[ Hewavitharanage et al[ : Phytochemistry 40 "0888# 24Ð30 30
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Angew[ Ent[\ 89\ 267[ 016[